The goal of this proposal is to elucidate the role of the middle-size T (mT) antigen in malignant transformation by polyoma virus. Polyoma virus encodes three related T antigens, all of which are implicated in transformation. The mT antigen, however, has been shown to be the most intimately involved, and may indeed be sufficient to induce most of the transformed phenotype. Employing an immunoaffinity technique which we have already tested successfully on a pilot scale, we propose to purify the mT antigen using antisera directed against synthetic peptides corresponding to unique region of the mT antigen. The mT antigen can be eluted from the immunoabsorbent in a native state with an excess of soluble peptide. Purified mT antigen will be tested for a variety of enzymatic and other activities which might be relevant to its function in transformation. Particular attention will be paid to the tyrosine protein kinase activity which is associated with mT antigen. The transforming proteins of a series of retroviruses are tyrosine protein kinases and aberrant tyrosine phosphorylation appears to play a role in transformation by these viruses. We aim to determine whether the tyrosine protein kinase associated with mT antigen is an intrinsic activity or whether it is due to a tightly interacting cellular enzyme through the use of a number of techniques including affinity labeling with nucleotide analogues. We have found that only a small fraction of the mT antigen has associated protein kinase activity. This form has an apparent molecular weight of 200,000 daltons compared to the bulk of mT antigen which has an apparent molecular weight of 50,000 daltons. This complex form of mT antigen will be characterized to see whether it contains a cellular tyrosine protein kinase. We will assess the significance of tyrosine phosphorylation in transformation by polyoma virus by comparison with cells transformed with viruses encoding tyrosine protein kinases. A large fraction of mT antigen is found affiliated with a membrane fraction of the cell. This location appears to be important for transformation. We propose to investigate the nature and topology of this membrane attachment using antibodies directed against specific epitopes of mT antigen. We hope these studies will contribute to an understanding of transformation by polyoma virus at a molecular level.